The botulinum neurotoxins (BoNTs) comprise a family of seven immunologically distinct proteins synthesized primarily by strains of the anaerobic bacteria, Clostridium botulinum. These toxins, designated A through G, are the most lethal substances known. For all clostridial neurotoxins, toxicity is produced in four stages: binding to ectoacceptors on the surface of motor nerve endings, internalization of the toxin-receptor complex into the cytoplasm, translocation of the toxin via a pH-dependent event and inhibition of impulse-evoked acetylcholine secretion via proteolytic cleavage of one of the SNARE proteins, ultimately leading to flaccid paralysis. For severe exposures, death ensues from paralysis of the diaphragm and intercostal muscles unless ventilatory support is rendered. Toosendanin is a triterpenoid derivative with a 4,4,8- trimethylfuranylsteroid skeleton that is an active ingredient extracted from the bark and fruit of plants in the family Melia, which has been used in traditional Chinese medicine. Over twenty years ago two reports published in Chinese journals described toosendanin as an effective antibotulinal compound in both monkey and mouse models. Disappointingly, these results have remained dormant, as no other laboratory has published complimentary findings to these initial reports. We have preformed preliminary studies on the natural product toosendanin using the "gold standard assay" for neutralization against the lethal effects of BoNT, a mouse model, and have found that indeed it can act as an antibotulinal small molecule agent. The current proposal is put forth as a platform from which we will elucidate the key features that are central to the antibotulinal properties of toosendanin. Hence, our specific goals are (1) To establish which chemical functionalities are necessary for the antibotulinal properties of toosendanin in the mouse using a semi-synthetic approach. (2) Our preliminary studies have uncovered the importance of the C14-C15 epoxide embedded within toosendanin as related to its biological activity. As such we have constructed a synthetic route to a scaffold that contains this moiety, as an analog to be investigated in animal models. (3) Toosendanin has an antibotulinal effect; however, it is unclear whether toosendanin itself or a metabolite of this molecule are responsible for its biological activity. To complement our semi-synthetic efforts we will investigate whether toosendanin's activity is due to a metabolite of its original structure; to accomplish this goal, liver microsomes and purified enzymes will be used and isolated products will be examined for antibotulinal effects. Botulinum neurotoxins (BoNTs) are the most lethal substances known, with potency approximately one million times greater than cobra toxin and one hundred billion times greater than cyanide. With the current warfare and terrorist activities around the world, in particular directed against the American people and infrastructure, a vastly enhanced defense against potential bioterrorist weapons is of extremely high priority. Our proposal aims to study toosendanin, a natural product previously used in traditional Chinese medicine, in an effort to determine the mechanism of its antibotulinal activity, with the hope of ultimately identifying new drugs for treating exposure to BoNTs. [unreadable] [unreadable] [unreadable]